Dopamine- 1-mediated Stimulation of Phospholipase C Activity in Rat ...

THEJOURNALOF BIOLOGICAL CHEMISTRY

Vol. 264, No. 15, Issue of May 25, pp. S739-8745,1989 Printed in U.S.A.

Dopamine- 1-mediated Stimulation of Phospholipase C Activity in Rat Renal Cortical Membranes* (Received for publication, April 20, 1988)

Christian C. FelderS, Melvin Blecher, and PedroA. Jose From the Departments of Pediatrics, Physiology and Biophysics, and Biochemistry, Georgetown University Medical Center, Washington, D. C. 20007 and Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland 20892

PhospholipaseC(PL-C)mediatestransduction of neurotransmitter signals across membranes via hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2),leading to generation of second messengers inositol- 1,4,5-trisphosphate and diacylglycerol. In this study, dopamine-1 (DA-1) but not dopamine-2(DA-2) agonists were shown to stimulate PL-C activity in renalcortical membranes. The DA-1agonist,SKF 82526, stimulated the release of inositol phosphates from renal cortical membranes prelabeled with [‘HI myoinositol. The majorityof the label(75%)was found in phosphatidylinositol followed by PIP2 (15%)and phosphatidylinositol-4-phosphate (10%).A DA-1 specific effect on PL-C activity wasalso observed in an in vitro assay of PL-C activity in renal cortical membranes and basolateral and brush border membranes using [‘H]PIP2 as the substrate. Dopamine and SKF 82526 stimulated the release of inositol phosphates fromadded [‘H]PIP2 in a concentration-dependent manner. This release wasblocked by the DA-1 antagonist SCH 23390 but not by the a-adrenergic antagonists phentolamine and prazosin. In contrast, theDA2 agonist LY 171555 had no effect on inositol phosphate release. Guanosine5’-(3-O-thio)triphosphateenhanced while guanyl-5’-yl thiophosphate attenuated the DA-1 agonist-stimulated PL-C activity. PL-C activity as measured by [‘H]PIP2 hydrolysis had a pH optimum of 6.5, was inhibited by Mg2+concentrations above 1mM, was linear with time and protein concentration, and was sensitive to phosphatidylserine and calcium concentrations. We conclude that PL-C is activated by DA-1 but not DA-2 agonists in renal cortical membranesas well as both the basolateral and brush border renal tubular membranes. It is speculated that this action may mediate the natriuretic effects of dopamine in renal tubular epithelia.

fied in renal membranes with phosphatidylinositol (PI)’ being the most predominant, followed by phosphatidylinositol-4,5biphosphate (PIP2)and phosphatidylinositol-4-phosphate (PIP) (1, 2). Hruska et al. (2) have identified the presence of diglyceride kinase, PI kinase, and PIP kinase in the renal brush border membrane. Phospholipase C (PL-C) hydrolysis of PI was reported in rat renal cortical and medullary slices (3) as well as in purified brush border membrane (4). More specifically, the activity of PL-C is enriched in the brush border membrane over crude plasma membranes for PI (PIP and PIPz were not tested). Tou et al. (5) have identified a renal cytosolic PL-C that hydrolyzes PIP and PIP2 but not PI. Diacylglycerol and inositol phosphates are released from [3H]inositol-labeled canine proximal tubular basolateral membrane upon stimulation with parathyroid hormone (6). Dopamine (DA) causes anatriuresis whichmay occur through the occupation of DA receptors of the DA-1 subtype (7-9). DA is synthesized in proximal renal tubules where DA receptors are located (10-12). However, the means by which DA receptor occupancy generates a signal which regulates sodium transport in the renal tubule is unclear. DA-1 receptors arelinked to adenylate cyclase stimulation inseveral cell types including renal epithelia (12, 13). A role for adenylate cyclase in the regulation of sodium transport in renal tubules has been suggested (14). Involvement of the inositol phospholipid cycle in the regulation of renal sodium transport has also been recently reported (15,16). An effect of DA-1 agonists on the phosphoinositide cycle has notbeen reported, however, there are conflicting reports on the role of DA-2 receptors on inositol phospholipid turnover in the anterior pituitary (1719). DA-2 receptors have also been linked to an increase in PI turnover inlight-activated retinal rod outer segments (20). A role for DA-1 or DA-2 receptors in renal membrane inositol phospholipid turnover has notbeen determined. The purpose, therefore, of this study was to determine the relationship between DA agonists and inositol phospholipid turnover in renal cortical membranes. EXPERIMENTALPROCEDURES

The study of the inositol phospholipid cycle in renal membranes is relatively new. Phosphoinositides have been identi* This work wassupported in part by Grants HL23081, HL 33498, and DK 39308 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solelyto indicate this fact. $ To whom correspondence should be addressed Dept. of Pediatrics, Georgetown University Hospital, 3800 Reservoir Rd., N. W. Washington, D.C. 20007. Tel.: 202-687-8675.

Materials-The following radiolabeled phosphoinositides and inositol phosphates were purchased from Du Pont-New England Nuphosphatidylinositol-4clear: phosphatidylinositol-4,5-bisphosphate, phosphate, phosphatidylinositol L-a, myo-inositol, D-inositol 1,4,5trisphosphate, D-inositol l,4-bisphosphate, D-inositol I-phosphate. Anion exchange resin (formate form) was purchased from Bio-Rad. Phosphatidyl serine was purchased from Sigma. GDPOSwas purl The abbreviations used are: PI, phosphatidylinositol; PIP, phosphatidylinositol-4-phosphate; PIP,, phosphatidylinositol-4,5-bisphosphate; PL-C, phospholipase C; DA, dopamine; GTPrS, guanoGDPPS, guanyl-5’-yl thiophosphate; sine 5’-(3-O-thio)triphosphate; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraaceticacid; PS, phosphatidylserine; IP, inositol-1-phosphate; IP,, inositol-1,4-bisphosphate; IPS, inositol-1,4,5-trisphosphate; PKC, protein kinase C.

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Dopamine Renaland

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chased from Boerhinger Mannheim. The following drugs were gifts: DA-1 agonist SKF 82526 (Smith Kline and French Laboratories, Philadelphia), DA-1 antagonist SCH 23390 (Schering Corporation, Kenilworth, NJ), DA-2 agonist LY 171555 (Lilly), phentolamine (Ciba Pharmaceuticals Inc.), and prazosin (Pfizer). Detection of Inositol Phospholipids by Thin Layer Chromatography-Detection and assessment of radiochemical purity of inositol phospholipids were determined by thin layer chromatography on Silica Gel60 plates (Merck) using the following solvent system: chloroform/methanol/20% methylamine in water, (12:7.2:2) (21). The organic layer from lipid-extracted samples or standards was dried in u m u o , and the residue was dissolved in 10 p1 of chloroform and spotted on thin layer chromatography plates. The developed plates were then sprayed with Enhance (Du Pont-New England Nuclear) and exposed for 2 weeks. The x-ray film was developed,and thedark areas of the samples were compared with known standards. Preparation of Renal Cortical Plasma Membranes-Crude plasma membranes were prepared by a modification of a previously described method (22). The renal cortex washomogenized with a Polytron homogenizer equipped with a PT-10probe (Brinkmann Instruments, Westbury, NY). This procedure was performed three times a t setting 6 for30 s with a 30-s cooling period between bursts. The crude homogenate was centrifuged (Beckman J21-B refrigerated centrifuge, 5-20 rotor) at 2,500 X g for 15 min at 0-4 "C. The supernatant was centrifuged for 20 min at 24,000 X g resulting in a pellet with two distinct membrane layers, a lower layer consisting primarily of mitochondria, and an upper layer consisting primarily of plasma membranes. The supernatant was discarded, and the membranes were swirled free of the lower, more densely packed layer in added PL-C assay buffer (23), (25 mM Tris-C1, 1.0 mM EGTA, 1.0 mMMgC12, 0.94 mM CaClZ, 5 mM LiCl, pH 7.4). The membranes were washed three times by resuspension in PL-C assay buffer, gentle homogenization with a glass Teflon homogenizer by hand for three passes, and centrifugation at 30,000 X g for 30 min. The final pellet was resuspended in PL-C assay buffer>o achieve a final protein concentration of 5 mg of $rotein/ml. This purification procedure resulted in a 5fold enrichment of the brush border and basolateral membrane markers y-glutamyl transpeptidase and Na'/K+ ATPase, respectively (22). Preparation of Renal Brush Border and Basolateral MembranesBasolateral and brush border membranes were prepared by a procedure developed by Liang and Sacktor (24) and Sheikh et al. (25). Purity was determined by the measurement of marker enzymes, and the results were similar to that previously reported (24). For brush border membranes, the brush border membrane marker y-glutamyl transpeptidase was enriched 15-foldcoincident with a 5-fold decrease in the basolateral membrane marker Na+/K+ATPase. For basolateral membranes, Na+/K+ ATPase was enriched 13.0-fold; y-glutamyl transpeptidase was slightly enriched to 2.2-fold indicating some contamination of brush border membrane in basolateral membranes. Two methods were used to assay PL-C activity. One method employed prelabeling endogenous inositol phospholipids in membrane fragments with my~-[~H]inositol and a second method which employed exogenouslyadded [3H]PIPz as the substrate. Assay of PL-C Activity in myo-PH]Inositol-labeled MembranesIn the first method, kidneys were removed from 4- to 5-month-old male Wistar Kyoto rats anesthetized with sodium pentobarbital. The outer two-thirds of the cortex was isolated, chopped into fine fragments with a razor blade, and placed in Dulbecco's modified Eagle's medium, pH 7.6 (GIBCO). The renal cortical fragments were rinsed several times with Dulbecco's modifiedEagle's medium to remove the blood and then suspended in Dulbecco's modified Eagle's medium (1 ml Dulbecco's modified Eagle's medium/g wet cortex weight) at 04 "C. The fragments were incubated with [3H]myo-inositol(100 pCi/ g wet cortex weight) (Du Pont-New England Nuclear) under humidified 95% Oz, 5% COZ atmosphere in a rotating water bath at 37 "C for up to 120 min. Maximum labeling occurred by 90 min (Fig. 1). Renal membranes were then prepared as described above and stored a t 0-4 'C for less than 1 h until theywere assayed for PL-C activity. PL-C activity was assayed by determining the release of inositol phosphates following the incubation of 200 p1 of membrane suspension (400-600 pg of protein) in PL-C assay buffer with or without the ligands or hormones to be studied (total volume of250 ~ 1 )The . reaction was initiated by the addition of the membrane suspension. At the end of the incubation period, the reaction was terminated by the addition of 1 ml of methanol/chloroform (2:1, v/v) followed by vigorous vortexing. Chloroform (0.5 ml) and 0.1 N HCl (0.5 ml) were added to each sample, vigorously vortexed, and incubated for 20 min at room temperature to extract the lipids. The samples were centri-

fuged at 1200 X g for 10 min to separate the water and lipid phases. The water-soluble inositol phosphates present in the upper aqueous phase and the inositol phospholipids present in the lower organic phase were subsequently analyzed as described below. To determine that endogenous inositol phospholipids were labeled by my~-[~H]inositol, membrane the lipids were extracted, deacylated, and theglycerophosphoinositide derivatives were separated by anion exchange chromatography (26). The eluted products were verified by deacylating authentic standard [3H]PI,[3H]PIP, and [3H]PIP2 under identical assay conditions (data not shown). To determine the relative abundance of individual water-soluble [3H]inositolphosphates released by the action of PL-C, the products were separated using a procedure of Berridge et al. (27). The products were verified by eluting authentic inositol phosphate standards. Assay of PL-C Activity Using Exogenously Added rH]PIPz os a Substrate-PL-C activity was also determined by using an exogenous source of [3H]PIPz as the primary substrate for PL-C andmonitoring the release of [3H]inositol phosphates. This assay was used in order to monitor the hydrolysis of PIP2 alone, independent of the hydrolysis of its natural precursors, PIP andPI. This determination would only be valid if there was no conversion of PIPz to either PIP or PI. In our studies, incubation of [3H]PIPzwith renal membranes for the standard incubation time of 10 min did not result in the formation of [3H]PIPor [3H]PI.Therefore, under theseconditions it is possible to monitor the hydrolysis of PIPZ, the main substrate of PL-C, without the simultaneous hydrolysis of its precursors. PL-C activity was linear with protein concentration over the range of 0.3-1.0 mg of protein/ ml. The in vitro assay of PL-C using added [3H]PIPz as a substrate required the presence of phosphatidylserine (PS) for activity. In the absence of PS, no significant PL-C activity was observed. The PL-C activity rose rapidly with increasing concentrations of PS and reached a plateau at 10 pg of PS/mg protein; no further increase in PL-C activity occurred up to 50pg of PS/mg protein (the highest P S concentration tested).P S concentrations greater than 10 pg/mg protein interferes with antagonist binding in renalmembranes, therefore, the PSconcentration was reduced to 6 pg/mg protein. Phosphatidylcholine and phosphatidylethanolamine had no effect on PL-Cactivity over the same concentration tested for PS. In preliminary studies, DA and DA-1 agonist SKF 82526 stimulated PL-C activity without added exogenous GTP. This apparent independence of DA-1 effect from guanine nucleotides could be due to the presence of endogenous or contaminatingGTP or reflect noninvolvement of G proteins in DA-1-mediated PL-C activation. These possibilities were examined by incubating rat renal plasma membranes with guanine nucleotides for 15 min prior to PL-C determination. [3H]PIPz(0.005 pCi/assay tube) and phosphatidylserine (6 pg/mg protein) were dried in vacuo to remove the organic solvents. The phospholipid residue was resuspended in sufficient PL-C assay buffer to provide 50 pl of emulsion/assay tube. The emulsion was prepared by sonicating the phospholipid mixture with aBranson Sonifier (Branson Instruments, Melville, NY) set at No. 50 for 15 s at 0-4 "C. The reaction mixture contained 150 pl of sonicated membranes (suspended in PL-C assay buffer, 100-200 pg of protein) and 50 pl of distilled water, with or without the ligands or hormones to be studied. Following a 5-min preincubation of the membranes and drugs at 37 "C in a Dubnoff shaking water bath, thereaction was started with the addition of 50 pl of phospholipid emulsion. The reaction was terminated after a10-min incubation period by the addition of a lipid extraction medium (1ml of methanol/chloroform, 2:1, v/v) followed by vigorous vortexing. Chloroform (0.5 ml) and distilled water (0.5 ml) were added to the samples and vortexed vigorously. Following a 20-min incubation at room temperature, the samples were centrifuged at 1200 X g for 10 min. One ml of the upper aqueous layer was added to 5 ml of scintillation fluid (Liquiscint, National Diagnostics) and the radioactive decaywas counted in a Beckman LS 1801 liquid scintillation spectrophotometer. Calculation of Free Calcium Concentrations-Equilibrium-free calcium concentrations were calculated based on the temperature, pH, concentration of EGTA, and calcium added to thebuffer (28). Experiments were performed in triplicate. Data are expressed as mean f standard error. Statistical analysis is described in figures and tables. RESULTS

DA-1-stimulated PL-C Activity-Two methods were used to measure PL-C activity. In the first method my~-[~H]ino-

Dopamine and Renal Phospholipase C Activity

a741

epinephrine ( M),also significantly stimulated the release of IP3 (20%) and IPz (16%) above basal levels but had no effect on IP release. The norepinephrine-induced release of IP3 and IPz wasblocked by the a-adrenergicantagonists phentolamine ( M) and prazosin (10" M) but not by the DA-1 antagonistSCH 23390 ( M ) (Table I). In the second method, PL-C activity was assayed by measuring the hydrolysis of exogenous added [3H]PIP2 to renal membranes in order to determine the effect of DA stimulation on PIPz hydrolysis independent of the hydrolysis of PI or PIP. PIPzis considered to be the primary substrate for PL-C resulting in the release of IP3 which subsequently stimulates intracellular calcium release (29,30). Therewas no significant metabolism of PIPz intoPIP or PI under the standard assay conditions (data not shown). Negligible conversion of IP to free inositol occurs with the inclusion of 5 mM LiCl to the incubation buffer limiting the amount of reincorporation of my~-[~H]inositol into PIor PIP. The specificity of DA-1stimulated PL-C activity for [3H]PI, [3H]PIP, and [3H]PIP2 was tested under identical assay conditions (data notshown). The DA-1 agonist SKF 82526 M) stimulated the hydrolysis of [3H]PIPz, but not [3H]PIPor [3H]PI, suggesting the presence of a PIPz-specific PL-C sensitive to DA-1 receptor occupation in renal cortical plasma membranes. Basal as well as DA-stimulated PL-C activity appeared to be linear for 30 min, while deoxycholate-stimulated PL-C activity was linear after a lag phase of about 10 min in crude renal cortical membranes (Fig. 2). Deoxycholate was included in these experiments as a positive control as it is known to stimulate PL-C activity directly (31). In brush border and basolateral membranes, PL-C activity appeared to be linear TIME. rnin. only in the first 15min and was lowerthan theactivity found FIG.1. Incorporation of rny~-[~H]inositol into kidney membrane inositolphospholipids. my~-[~H]Inositol was incubated with in cortical plasma membranes (Fig. 3). This may reflect the more extensive processing required to purify brush border and kidney cortical fragments for the times indicated, and plasmamem-

sitol was incubated with renal cortical fragments, and plasma membranes were subsequently prepared from the labeled fragments. The amount of my~-[~H]inositol incorporated into the renal membranes was measured by anion exchange separation of the deacylated glycerophospho derivatives. The majority of the label (75%) was found in PI followed by PIPz (15%) and PIP (10%) with maximum labeling achieved by 90 min (Fig. 1). Similar results have been shown in liver (29) and renal cortical plasma membranes (3). The release of water-soluble [3H]inositol phosphates from the endogenously labeled inositol phospholipids was monitored as anindex of PL-C activity. The DA-1 agonist SKF 82526 M ) significantly stimulated the release of IP3 (22%) and IPz (15%) over basal levels, but had no effect on IP release (Table I). The DA-1 agonist-stimulated release of IP3 and IPzwas blocked by the DA-1 antagonistSCH 23390 M ) but not by the q 2 adrenergic antagonist, phentolamine M ) or the a1antagonist, and prazosin (Table I). The a-adrenergic agonist, nor-

branes were prepared from the labeled fragments. The labeled inositol phospholipids were extracted and thedeacylated glycerophosphoinositide derivatives separated by anion exchange chromatography by eluting in a step wise ammonium formate gradient. Results are the mean f S.E. of three experiments performed in triplicate. Where no error bar occurs, the S.E. was smaller than thesymbol.

z 6000

a

TABLE I a Effect of DA-1 and a-adrenergic agonist and antagonists 0 2000 on inositol phosphate release from renal cortical plasma membranes prelabeled with myo-~Hlinositol I DA-1 agonist SKF 82526 or a-adrenergic agonist norepinephrine 0 10 20 30 40 (NE) alone increased therelease of inositol 1,4,5-trisphosphate (IPS) TIME, rnin. and inositol 4,5-bisphosphate(IPz)but not inositol 1-phosphate. The DA-1 stimulatedIP3 and IP2 release was blocked by the DA-1 antagFIG. 2. Time-dependent ['HIPIP, hydrolysis in rat renal onist SCH 23390 but not by the al-*-adrenergic antagonist phentol- membrane vesicles. Results are the mean f S.E. of three experiamineortheal-adrenergicantagonist prazosin. The stimulatory mentsperformedintriplicate. DOC, deoxycholate, a nonspecific effect of NE on inositol phosphate release was blocked by the astimulator of PLC activity. adrenergic antagonists but notby DA-1 antagonist showing specificity. Results (cpm/mg protein) are the mean f S.E. of three experiments Derforrned in tridicate. Experimental conditions

Basal SKF 82526 M SKF 82526 M + SCH 23390 M SKF 82526 M + phentolamine lo-' M SKF 82526 M + prazosin M NE 10-5 M + SCH 23390 M NE NE M + phentolamine lo-' M NE M Drazosin M

IP,

IP,

IP

144 f 5 436 f 14 1546 f 47 184 f 4" 501 f 12" 1465 f 43 144 f 5 419 f 16 1469 f 39 169 & 4" 475 1?: 20" 1497 f 34

173 f 5" 505 f 13" 1494 f 40 171 +. 5" 497 f 12" 1453 f 38 144 f 6448 f 20 1490 f 38 156 f 6 439 f 11 1498 +. 39 " p < 0.05 compared with basal, analysis of variance, and Dunnett's test.

+

I-

0

(L

a

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I

a

0

175 f 7" 473 f 10" 1451 f 29

1

z W

'oool 500

0I .

0

1'0

20 TIME, rnin.

30

40

FIG.3. Time-dependent ['HIPIP* hydrolysis in basolateral (BLMV)or brush border membrane vesicles (BBMV).Results are the meanof two experiments performed in triplicate.

Dopamine Renaland

8742

Phospholipase C Activity

basolateral membranes. The optimized PL-C assay was used to determine if either DA-1 or DA-2 receptor linked PL-C activity was present in kidney plasma membranes (Fig. 4). Both DA and the DA-1 agonist SKF 82526 stimulated PL-C activity (EC50 for DA = 1.02 f 0.03 X M and for SKF 82526 = 1.61 k 0.01 X 10" M). Concentrations of the DA-1 agonist SKF 82526above 100 p~ inhibited PL-C activity. The DA-2 agonist LY 171555 did not have any effect on basal PL-C activity. In addition, LY 171555 M) had no effect on SKF M)-stimulated PL-C activity (datanot shown). The DA-1 antagonistSCH 23390 M) partially blocked the DA-1 agonist SKF 82526 M)-stimulated PL-C activity (Fig. 5 ) . (In the studies with endogenously labeled inositol phospholipids, M SCH 23390 completely inhibited the DA-1-stimulated PL-C activity. It is possible that the phosphatidylserine, which is necessary for PL-C activation, interfered with the antagonist effect since it also affected the specific binding of DA-1 antagonist radioligand to renal plasma membranes (data not shown)). The&-adrenergican2500 A-8

SKF 112528

0-0

LY 171555

tagonists phentolamine ( W 4 M) and prazosin M) had no effect on DA-1 stimulated PL-C activity (Fig. 5). The different products released from PIPz during the PLC assay were separated by anion exchange chromatography. The primary product released was [3H]IP3 with minor amounts of the other inositol phosphates (Fig. 6). This was expected since the only radiolabeled substrate present was [3H]PIPz. The minor amount of IPz present may have occurred from the hydrolysis of I&. The IP2 could rapidly convert to IP which accumulates due to theinclusion of 5 mM LiCl in the incubation buffer. Sensitivity of PL-C Activity to Calcium and MagnesiumThe effect of increasing free calcium concentrations on basal and DA-stimulated (1 X M ) PL-C activity is shown in Fig.7.Below 1 X low7M and above 1 X M free calcium concentration therewas a marked inhibition of PL-C activity. Basal activity rose steadily over the range of 1 X M to 1 X M and was inhibited above 1 X M (EC50 for free calcium = 1 X M for basal activity). The DA-stimulated PL-C activity increased markedly above 1 X M was inhibited above 1 X M (EC50 for free calcium = 1 X M for DA-stimulated PL-C activity). Kidney membrane basal and DA-1 agonist SKF 82526-stimulated M) PL-C ac-

Y===I

loooo STANDARD IP

log[AGONIST],

M

b

5000t

FIG. 4. Effect of DA-1 and DA-2 agonists on the hydrolysis of added exogenous ['HIPIP2 in rat plasma membranes. The hydrolysis of added exogenous [3H]PIP2was measured as theappearance of water-soluble inositol phosphates, determined when DA, the DA-1 agonist SKF 82526, and the DA-2 agonist LY 171555 were added to the assay mixture a t the concentrations indicated. The results are the mean & S.E. for threeexperiments performed in triplicate.

0

300

if

200 100

'0

FIG. 5. Effect of DA-1 and a-adrenergic antagonists on DA1-stimulated ['HIPIP2 hydrolysis in rat renal plasma membranes. The hydrolysis of exogenously added [3H]PIP2was measured as theappearance of water-soluble inositol phosphates. Ligands were added in the following concentrations: DA-1 agonist SKF 82526 (lo-' M),DA-1 antagonist SCH 23390 M), al.2-adrenergic antagonist, M). phentolamine M), al-adrenergic antagonist, prazosin Results are the mean k S.E. of three experiments performed in triplicate.

10

20 30 40 FRACTIONS. 1 ml

50

FIG. 6. Separation of inositol phosphate products released from the hydrolysis of added exogenous ['HIPIP2 to rat renal plasma membranes. The elution profiles of inositol phosphate standards separated by anion exchange chromatography by the addition of a step-wise ammonium formate gradient are shown in the top three panels. Free inositol (ZNOS) and glycerophosphoinositide (GPZ) were first eluted with 5 ml each of 5 mM inositol, 5 mM disodium tetraborate, and 60 mM sodium formate, respectively. This was followed bythe addition of 10 ml of 100 mM formic acid, 200 mM 10 ml of ammonium formate which eluted inositol 1-phosphate (ZP), 100 mM formic acid, 400 mM ammonium formate which eluted inositol-1,4-bisphosphate (ZPZ),and finally 10 ml of 200 mM formic acid, 1 M ammonium formate which eluted inositol-l,4,5-trisphosphate (ZP3).The bottom panel shows the products released when rat renal cortical membranes are preincubated in the absence (basal) or presM). Results are representence of the DA-1 agonist SKF 82526 ative of three experiments.

8743

Dopamine and Renal Phospholipase C Activity 2600

-

P8

1500

-

E

1000

-

600

-

2000

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+ SKF a2521 (10-sy) 2000

2

a

8

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0

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-b

-?

-6

LOG

I

I

-3

@ d + J FREE.

-1

BlsAL

FIG. 7. Effect of varying free calcium concentrations on the hydrolysis of added exogenous ['HIPIP2 in rat renal plasma measured membranes. The hydrolysis of added exogenous [3H]PIP~ as the appearance of water-soluble inositol phosphates was determined over a range of free calcium concentrations for both basal and DA (lo-* M)-stimulated PL-C activity. Free calcium was calculated from the equilibrium of an EGTA-buffered system (see "Experimental Procedures" section for details). The results are the mean & S.E. of three experiments performed in triplicate. The ECS0for basal and DA-stimulated PL-C activity was estimated to be 1 PM and 100 nM, respectively.

-5 -4 LOG[MQH], M

-3

-3

M

M) was PIP2 in the presence of DA-1 agonist, SKF 82526 inhibited byGDPj3S in a concentration-dependent manner. The results are the mean k S.E. of three experiments performed in triplicate. When no error bars occur, the S.E. was smaller than the symbol. Significant changes from control ( p < 0.05, Dunnett's test) occurred only when the GDPj3S concentration was greater than

*

-2

FIG. 8. Effect of varying magnesium concentrations on the hydrolysis of added exogenous ['HIPIP* in rat renal plasma membranes. The hydrolysis of added exogenous [3H]PIPzmeasured as the appearance of water-soluble inositol phosphates was determined over a range of M e concentrations. The results arethe mean of triplicate determinations and are representative of two experiments. The ICsofor basal- and SKF 82526-stimulated PL-C activity was estimated to be 1.1and 1.3 mM, respectively.

tivity was inhibited by magnesium concentrations above 5 X M (Fig. 8).Bothbasaland SKF 82526 M)-stimulated PL-C activity had a pH optimum of 6.5. Effect of Guanine Nucleotide on DA-1 -stimulated PL-C Actiuity-GDPPS inhibited DA-1-stimulated PLC activity in a concentration-dependent manner.GDPPS by itself minimally inhibitedbasalPL-C activity (Fig. 9). GTPySandGTP increased PL-C activity at lop4and M, respectively. The M) increased the ability of DA-1 addition of GTPyS agonist, SKF 82526 M ) , to stimulatePL-C activity (Fig. 10). Preparingthe membranes in 1.25 mM ATP and 1.56 mM MgC12 did not increase the sensitivity to guanine nucleotide effect (32). Multiple membrane washings, cholate treatment (33),and preincubation of membranes with dopamine to deplete any endogenous GTP did not alter the effects of either GDPPS or GTPrS (data not shown). DISCUSSION

The presentstudies suggest thatthe DA-1 receptor is associated with stimulation of PL-C activity in renal cortical

*#

1.

1 -6

-4

LOG [GDP-beta-SI,

FIG. 9. Effect of varying concentrationsof GDPBSon basal-

I

0

-5

-1

and DA-1-stimulated PL-C activity using exogenous ['HIPIPp in rat renal plasma membranes. The hydrolysis of added ['HI

M

I C

n

30001 1000

H

0

o"o."O BASAL

I1 1 *

1

FIG. 10. Effect of varying concentrations of GTP on basal and GTPrS on basal- and DA-1-stimulated PL-C activity using exogenous ['HIPIP2 in rat renal plasma membranes. The hydrolysis of added [3H]PIP2in the presence and absence of DA-1 t M. The agonist, SKF 82526 (lo-' M) was increased by G T P r S a lo-' hydrolysis of added [3H]PIP2was increased in the presence of GTP M) (*p < 0.05 versus basal, #p < 0.05 versus DA-1, analysis of variance, Newman-Keuls test). The results are the mean k S.E. of three experiments performed in triplicate.

membranes as well as renal cortical basolateral and brush border membranes. DA-1-stimulated PL-C activity was demonstrated in the following two ways: the release of inositol phosphates from my~-[~H]inositol prelabeled cortical membranes, and the release of inositol phosphates from the hydrolysis of exogenous added [3H]PIP2 in renalcortical membranes. The release of inositol phosphates from membranes prelabeled with my~-[~H]inositol was significantly increased by a DA-1 agonist and an a-adrenergic agonist. The increase in released inositol phosphates occurred in theIP3and IP2 fraction without affecting IP. Presumably the IP2was generated from the metabolism of IPS and notfrom the hydrolysis of PIP since DA-1 agonist did not affect the hydrolysis of exogenously added [3H]PIP. The DA-1-stimulated inositol phosphate release was blocked by a DA-1 antagonist but not by the nonselective a-adrenergic antagonist, phentolamine or the al-selective antagonist, prazosin; norepinephrine-stimulated inositol phosphate release was blocked by a-adrenergic antagonistsbutnot by a DA-1 antagonist.Theseresults suggest that both DA-1- and a-adrenoceptor-stimulated PL-

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C activities are present in renal cortical membranes. Further support for a DA-1-stimulated PL-C activity in renal membranes was shown in the experiments where [3H] PIP' was the exogenous source of substrate in the presence of calcium and phosphatidyl serine. DA-1 stimulated PL-C activity was blocked by a selective DA-1 antagonist but not by a-adrenergic antagonists. There was no effect of a DA-2 agonist on PL-C activity although DA-2 receptors are present in these membranes.' Theseresultsare analogous to our previous report that DA-1 agonists increased renal cortical adenylate cyclase activity while DA-2 agonists were without effect (12, 34). While DA-2 receptors may be associated with a decrease in phosphatidylinositol turnover in anterior pituitary (17, 181, the present report provides the first evidence linking the DA-1 receptor to stimulation of PL-C activity. The PL-C activity measured in renal cortical membranes exhibited a calcium, magnesium, and PS sensitivity. PL-C activity was inhibited below100 nM and above 1 mM free calcium concentration. Magnesium concentrations above 500 PM were inhibitory. PL-C activity had an optimum requirement for PS which couldnot be replaced by phosphatidylcholine or phosphatidylethanolamine. Using exogenous [3H]PIPz as the substrate, the renal plasma membrane PL-C has characteristics similar to that described by Majerus et d. (29) in platelets. Other investigators have described the presence of PL-C activity in renal plasma membranes (2-4) but not the linkage to DA-1 receptors. We have not determined which isomeric form of PL-C is present in the renal cortex or which form is coupled to theDA-1 receptor. G proteinslinked to membrane-associated effector systems that generate second messengers have been demonstrated in many cells including the kidney (35). In the current experiments DA-1 and a-adrenergic stimulation of PL-C activity occurred in the absence of added GTP. Thus, there is the possibility that G proteins are not involved in PL-C activation. Indeed stimulation of phosphoinositide hydrolysis by aladrenoceptors may be associated with G protein-dependent and -independent mechanisms (36). It is more likely however that the apparent independence of DA-1-mediated PL-C activation from guanine nucleotides is due to the presence of residual endogenous or contaminating GTP since GDPPS inhibited the DA-1 stimulated PL-Cactivity. The presence of endogenous GTP may also explain the high concentrations of GTPyS needed to elicit DA-1-stimulated PL-C activity (37). PL-C linkage to G proteins hasbeen reported to be facilitated by preparing platelet membranes in MgClz and ATP (32) or cholate treatment of plasma membranes (33). No such beneficial effects were noted in our studies. Multiple washings and pretreatment of membranes with dopamine to deplete endogenous GTP also did not alter the GTPyS and GDPPS effects. Dopamine has been reported to increase GTP levels in rat retina (38) and may make GTP depletion difficult. Fain et al. (39) reviewed the difficulties in showing guanine nucleotide effects on hormone activation of PL-C using membranes and exogenous PIPz (39). However, in our studies micromolar concentrations of GTPyS were still needed even when phospholipase C activity was measured in membranes prelabeled with [3H]inositol(data notshown). Even though a nonspecific effect of guanine nucleotide is possible, the combined results of our studies using GDPpS andGTPyS suggest that theDA1-receptor-PL-C linkage is guanine nucleotide dependent. Occupation of the DA-1 receptor can stimulate both PL-C activity shown in these studies andadenylate cyclase activity in renal cortical homogenates as well as brush border and C. C. Felder, M. Blecher, and P. A. Jose, unpublished observations.

basolateral membranes.' The question of whether the same DA-1 receptor couples to both adenylate cyclase and PL-C effector systems or whether multiple forms of the DA-1 receptor are present in these membranes remains to be answered. In the brain the binding of the DA-1 antagonist lZ5I-SCH 23982, the iodinated form of SCH 23390, suggests multiple sites (40). Wehave reported that DA-1 agonist stimulates adenylate cyclase activity independent of PL-C activity in renal cortical membranes (37). The role of phosphatidylinositol metabolism and dopamine effects in the kidney remains to be determined. DA decreases proximal tubular solute and fluid transport and stimulates both adenylate cyclase and PL-C activities similar to the actions reported for parathormone (41-43). Cyclic AMP has been shown to inhibit Na+/H+ antiport, Na+/Pi symport in proximal tubular brush border membranes and increase luminal backflux at paracellular pathways (44). Thus, a DA-1 related increase in cAMP (34) can explain the inhibitory effects ofDA on sodium transport in the proximal tubule. However, Chan et al. (45) reported that DA inhibition of proximal tubular fluid and HCO; transport isseen at concentrations that increase cytosolic calcium but not cAMP production. This inhibitory effect of DA in rat proximal tubule perfused in vivo could be mimicked by the calcium ionophore A23187, with no additive effect when both DA and A23187 are used. Moreover, removal of calcium from the perfusate blunts the effect ofDA (45). Thus, changes in intracellular calcium induced by DA can influence proximal transport (independent of CAMP). PL-C catalyzes the breakdown of inositol phospholipids, notably PIP2,with the production of inositol phosphates ( e g . IP3) and sn-l,Z-diacylglycero1(46,47). IPS (or related IPS, e.g. cyclic IPS) has been demonstrated to increase intracellular calcium while sn-1,2-diacylglycerolactivates protein kinase C (PKC). We suggest that DA may exert its effects in part via the PL-C system. However, it is unlikely that DA-1-stimulated PIP' turnover is solely responsible for the natriuretic effect of DA. al-adrenergic agonists which mimick the DA-1related increase in PL-C activity (46,47) increase (48) rather than decrease Na+ absorption. PKC increases Na+/H+ antiport in proximal tubular cells (15), the opposite of the DA effect (7, 8). (In some cells PKC actually inhibits Na+ transport (16).) Moreover, DA increases cytosolic calcium transiently (45) while al-adrenergic agonists produce a sustained increase in intracellular calcium (49). Since neither adenylate cyclase nor PL-C activation individually explains the effects of DA on proximal tubular transport, we propose asynarchial system for both adenylate cyclase and PL-C similar to theaction of parathormone (49). Parathormoneand DA-1 agonists have similar effects on proximal tubular transport and both stimulateadenylate cyclase and PL-C (42, 43), suggesting similar mechanisms of action. How DA-1 agonists or parathormone decreases Na+ transport (via adenylate cyclase and CAMP, and via PL-C, IP3,and calcium) without exhibiting the Na+ stimulatory effect of sn-1,2-DAG via PKC is not known. It has been suggested that the relative amounts of sn-1,2-DAG and IP3 formed during hormonal stimulation of cells may beimportant for the expression of the separate signaling roles of these compounds because of the different sensitivities of their effect (46). Thus, a relatively mild agonist-induced stimulation of PL-C activity may produce enough IPS to elicit mobilization of calcium but not enough sn-1,2-diacylglycerol to activate PKC. This hypothesis would explain the increase in Na+ reabsorption due to al-adrenergic agonists and the decreased Na+ reabsorption due to DA-1 agonists, assuming that the


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former increases both IP3 and PKC activity, and the latter increases IPS and notPKC.